A time course of NADPH-oxidase up-regulation and endothelial nitric oxide synthase activation in the hippocampus following neurotrauma

Nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase; NOX) is a complex enzyme responsible for increased levels of reactive oxygen species (ROS), superoxide (O-2(center dot-)). NOX-derived O-2(center dot-) is a key player in oxidative stress and inflammation-mediated multiple secondary injury cascades (SIC) following traumatic brain injury (TBI). The O-2(center dot-) reacts with nitric oxide (NO), produces various reactive nitrogen species (RNS), and contributes to apoptotic cell death. Following a unilateral cortical contusion, young adult rats were killed at various times postinjury (1, 3, 6, 12, 24, 48, 72, and 96 h). Fresh tissue from the hippocampus was analyzed for NOX activity, and level of O-2(center dot-). In addition we evaluated the translocation of cytosolic NOX proteins (p67(Phox), p47(Phox), and p40(Phox)) to the membrane, along with total NO and the activation (phosphorylation) of endothelial nitric oxide synthase (p-eNOS). Results show that both enzymes and levels of O-2(center dot-) and NO have time-dependent injury effects in the hippocampus. Translocation of cytosolic NOX proteins into membrane, NOX activity, and O-2(center dot-) were also increased in a time-dependent fashion. Both NOX activity and Or were increased at 6 h. Levels of p-eNOS increased within 1 h, with significant elevation of NO at 12 h post-TBI. Levels of NO failed to show a significant association with p-eNOS, but did associate with O-2(center dot-). NOX up-regulation strongly associated with both the levels of O-2(center dot-) and the total NO. The initial 12 h post-TBI are very important as a possible window of opportunity to interrupt SIC. It may be important to selectively target the translocation of cytosolic subunits for the modulation of NOX function. (C) 2014 Elsevier Inc. All rights reserved.